Thrombotic disorders afflict a large number of people. Nearly 576,000 new cases of deep vein thrombosis and pulmonary embolism, two of the most common thrombotic conditions, are diagnosed every year in the US alone. Even more alarming is that thrombotic disorders are ~3-fold more likely in people with cancer. Anticoagulants, including heparins and coumarins, are the mainstay of treatment and prevention of thromboembolic disorders. Yet, the current anticoagulation therapy is beset with a significant number of adverse reactions including enhanced bleeding risk, immunological reaction, genetic variation in metabolism, food or drug interactions and liver toxicity. In addition, problems such as patient-to-patient response variability, narrow therapeutic index, inadequate duration of action, poor oral bioavailability, the need for frequent coagulation monitoring, and high cost to benefit ratio further complicate the treatment of thrombotic conditions. We reasoned that to reduce the problems associated with the current anticoagulation therapy, molecules radically different from all the current agents (heparins, warfarins, hirudins, and peptidomimetics) should be discovered. We have discovered that chemo-enzymatically synthesized lignins, represented by three sulfated dehydropolymer (DHP) molecules, named CDs, FDs and SDs, possess extremely interesting anticoagulant properties and a novel mechanism of action. 1) Sulfated DHPs (CDs, FDs and SDs) prolong prothrombin time at concentrations 2-6-fold below that of the clinically used LMWH enoxaparin, while in the activated partial thromboplastin time assay they required 2-6-fold higher concentration. 2) Whole blood clotting studies using thromboelastography and hemostasis analysis system reveal that our novel anticoagulants inhibit clotting with potency only 18-30-fold weaker than enoxaparin. 3) Mechanistically, the new molecules inhibit thrombin, factor Xa and factor XIa with IC50 values in the range of 10-240 nM. 4) In contrast, they inhibit factor IXa and factor VIIa with IC50 values 60-170-fold and >840-fold higher, respectively suggesting high selectivity for thrombin and factor Xa. 5) This potent inhibition arises primarily from direct inhibition of thrombin and factor Xa, although indirect inhibition mediated by antithrombin may also contribute. 6) Direct inhibition arises from an allosteric disruption of thrombin's catalytic apparatus (reduction in kCAT). 7) Competitive binding studies suggest that CDs interacts with exosite II of thrombin, a site not typically associated with inhibition. 8) A chemically synthesized CDs-based monomer inhibits thrombin and factor Xa with an IC50 of ~30 <M. 9) Studies using A549 lung and HepG2 liver cell lines show no induction of toxicity by CDs, FDs, and 2-5MSC at concentrations as high as 50 mg/L. Thus overall, the phenomenon of potent anticoagulant activity (in vitro, plasma and whole blood) by CDs, FDs and SDs, complemented by a novel molecular mechanism of action and a radically different structural scaffold from each of the current anticoagulants, raises a strong possibilities for discovering novel anticoagulants. We propose to 1) investigate the interaction of sulfated DHPs and DHP-based synthetic molecules with proteins (factors IIa, Xa, XIa, XIIa, XIIIa, IXa, VIIa, antithrombin, kallikrein, activated protein C plasmin, trypsin, etc.) at a molecular level; 2) perform combinatorial virtual library screening of CDs, FDs and SDs sequences binding to thrombin, factor Xa and antithrombin to identify `needle(s) in the haystack'; and 3) chemically synthesize specific homogeneous molecules, study their cellular toxicity, and measure their anticoagulation potential in vitro, ex vivo and in vivo. PUBLIC HEALTH RELEVANCE: Nearly 576000 new cases of deep vein thrombosis and pulmonary embolism, two of the most common thrombotic conditions, are diagnosed every year in the US alone. Thrombotic disorders are even more prevalent in people with cancer. The proposed research on novel dual direct inhibitors of thrombin and factor Xa aims to improve current anticoagulation therapy, which is beset with significant number of adverse reactions and limitations.